Comammox Microorganisms' Contribution to Nitrification in Soil – COMICONS

Soil sampling
Microcosm experiments will be first established with nitrification kinetics, physicochemical properties and microbial communities profiled. We will use the SOERE-PRO (environmental research monitoring and experimentation systems) network that was established to determine the effect of different applications of organic residues (sludge, manure, compost). Microcosms will be established to determine whether comammox bacteria can utilise, and compete with, AOA and AOB for both inorganic and organic ammonium sources.

Cultivation of comammox bacteria.
Enrichment cultures (with the aim to achieve eventual isolation) will be established using minimal salt media based on those used for cultivating aquatic comammox bacteria.

High throughput DNA sequencing.
Sequence analysis of PCR amplicons and genomic sequences will be performed using the in-house sequencing platform at Ampère to profile comammox communities. For genome sequencing of comammox isolates, genomic DNA will be isolated and sequenced using both the Illumina MiSeq and Oxford Nanopore MinION. In our experience the analysis of sequencing runs from both platforms facilitates the production of complete or near-closed draft genomes.

Mesocosms and N2O measurement
Soil mesocosms will be established using 5 soils and N2O emissions will be determined using a novel autonomous gas analysis system for soil microcosms. Soil microcosms with be established and incubated with different nitrification inhibitors to modulate the growth of different nitrifier groups.

Completed results this far

Using a model agricultural soil, our recent work has demonstrated comammox Nitrospira clade B can be more abundant than other AO communities at near-neutral pH soil where they possess ecological niches distinct from canonical ammonia oxidising archaea (AOA) and bacteria (AOB). Specifically, whereas AOA and AOB grew in aerated soil (~60% water filled pore space) using mineralised organic N (AOA) or added inorganic ammonium (AOA and AOB), comammox Nitrospira only grew using ammonia derived from mineralised organic N at higher water contents, indicating that oxygen availability may be a factor controlling their activity. To test the hypothesis that comammox Nitrospira activity increases towards microaerophilic conditions, microcosms were established with increasing water filled pore space or decreasing headspace oxygen concentration. Specifically, pH 7.5 soil was incubated for 30 days with 12C- or 13C-CO2 at 30%, 40% or 50% soil water content (w/w) with an initial 21% headspace oxygen concentration, or with an initial 21%, 10.5% or 2.1% headspace oxygen concentration and a 30% water content. DNA-SIP combined with quantitative PCR of group-specific amoA genes was used to assess AOA, AOB and comammox growth and activity. AOA and AOB were active and inactive, respectively, in all conditions, but comammox Nitrospira demonstrated growth and incorporation of 13C only at 40% water content or at 2.1% headspace oxygen. These results demonstrate that reduced oxygen availability within the soil matrix may be an important factor in controlling Nitrospira comammox activity.

We have made significant progress in understanding the conditions when comammox bacteria are active in soil. We anticipate that comammox bacteria will not be major contributors to fertiliser loss in conventional agricultural systems. However, in natural un-managed soils systems (which represent the largest proportion of the Earth's surface), they may make major contributions to nitrogen cycling and nitrous oxide production.

By undertsanding the contributions of different ammonia oxidiser groups (AOA, AOB and comammox) the project has potential to provide economic benefits, through more efficient use of N-based fertilisers, and environmental benefits, improving the quality of life through reductions in ammonia and nitrous oxide emissions to the atmosphere and reductions in nitrate levels in groundwater and rivers.

Two conference presentations and one manuscript in preparation

Transformation of reactive nitrogen (N) in the environment arguably represents the most anthropogenically impacted elemental biogeochemical cycle on Earth. Input of N via fertilisers (100 Tg/y) and deposition of atmospheric N (25 Tg/y) now exceeds that entering naturally through nitrogen fixation (110 Tg/y) and fertiliser input is predicted to increase with an increasing global population. A key process of nitrogen conversion in the environment is the process of nitrification whereby ammonia is oxidised to nitrate via nitrite. Nitrite rarely accumulates in soil and ammonia oxidation usually limits nitrification rates. While ammonium is retained in the soil, nitrate is highly mobile and ammonia oxidation leads to an estimated loss of 67% of applied fertiliser and a global economic cost of $15.9 billion. As well as resulting in deleterious environmental consequences in terrestrial and aquatic systems, this dramatic increase in reactive nitrogen addition has concomitantly increased emissions of the greenhouse gas nitrous oxide (N2O), which contributes to global warming and stratospheric ozone depletion.

This microbially-mediated process is traditionally considered to be performed by two physiologically distinct groups, with ammonia converted to nitrite by ammonia oxidisers (AO), and nitrite to nitrate by nitrite oxidising bacteria (NOB). Until recently, two major groups of AO, ammonia oxidising archaea (AOA) and ammonia oxidising bacteria (AOB), were assumed to be the major contributors to aerobic ammonia oxidation in soil, using ammonia as their only source of energy. Over the last decade, considerable research effort has demonstrated niche differentiation both within and between AOA and AOB communities, and different relative contributions to nitrogen fertiliser loss and associated pollution. However, in 2015, two paradigm-shifting studies revealed that bacteria belonging to the phylum Nitrospira are not only also capable of ammonia oxidation, but are indeed capable of complete ammonia oxidation to nitrate, or ‘comammox’. This finding was surprising as all previously cultivated Nitrospira strains were strictly NOB, i.e. they could only perform the second step of nitrification. Importantly, the abundance of Nitrospira communities in many environments, including soil, are typically greater than that of either AOA or AOB, indicating that they may make the major contribution to ammonia oxidation in soil. There is therefore an urgent requirement to determine their functional importance in soils and whether they make major contributions to fertiliser-associated N loss and pollution.

COMICONS proposes a multidisciplinary approach combining expertise in microbiology, molecular ecology, genomics, biogeochemistry and modelling to characterize the physiology, ecological niche(s) of comammox bacteria and their overall contribution to nitrogen cycling processes in soil. Specifically, we will determine under what conditions comammox bacteria are active in soil and what sources of N they utilize for growth. We will characterize their activity in situ in the soil environment, together with detailed physiological analysis of novel strains obtained in laboratory culture. Importantly, we also model their contribution to N transformations in soil with respect to fertiliser addition, determine their contribution to nitrous oxide emissions in soil, and determine whether they have distinct ecological niche(s) in comparison to other characterized groups of ammonia oxidisers. Overall, this research will lead to a fundamental breakthrough into understanding of who are the major contributors to nitrogen cycling, informing future approaches that aim to mitigate nitrification-associated pollution.